Type I diabetes is caused by the autoimmune destruction of the pancreatic islet insulin-producing beta cells. Insulin administration does not prevent the long-term complications of the disease, since the optimal insulin dosage is difficult to adjust. Replacement of the damaged cells with regulated insulin-producing cells is considered the ultimate cure for type 1 diabetes. Pancreas transplantation has been successful but is severely limited by the shortage of donors. With the development of new islet isolation and immunosuppression procedures, significant success has been reported using islets from 2-3 donors per recipient (Shapiro A M, Lakey J R, Ryan E A et al. New Engl J Med 2000; 343:230-238). This progress underscores the urgent need for developing alternatives to human pancreas donors, namely abundant sources of cultured human 13 cells for transplantation.
Terminally differentiated, postmitotic islet cells are difficult to expand in tissue culture. Adult and fetal human islet cells grown on HTB-9 matrix in RPMI 1640 medium containing 11 mM glucose, and supplemented with 10% FBS and hepatocyte growth factor, were shown to proliferate at the most for 10-15 population doublings, after which they underwent senescence. The replication span could not be extended by expression of the catalytic subunit of human telomerase (hTERT), which was introduced into the cells with a retrovirus (Halvorsen T L, Beattie G M, Lopez A D, Hayek A, Levine F. J Endocrinol 2000; 166:103-109). Due to massive cell death, this method resulted in a 3-4 expansion of the islet cell mass.
The process of beta cell expansion by prolonged culture is accompanied by dedifferenetiation of the cells.
In many instances, the dedifferentiation of cells is accompanied by drastic changes in phenotype in which their morphology changes from that of epithelial cells containing extensive cell-cell junctions and cytokeratin filament networks, to cells with a fibroblast or mensenchymal appearance. This process of dedifferentiation is known as an epithelial to mesenchymal transition (EMT) and is believed to be mediated in part by the induction of the zinc finger transcription factor Snail.
Slug, a Snail family member, has been implicated in the re-epitheliazation of cutaneous wounds, as well as in the regeneration of damaged skeletal muscle after damage. In the area of pancreatic stem cells, Gershengorn et al [Science, 2004, 306, 2261-2264], suggested that cultured primary beta cells are capable of EMT and dedifferentiation into a fibroblastoid-like cell type. Gershengorn suggested that this process could be reversed, and these fibroblastoid cells could then be redifferentiated into pancreatic endocrine cells by a so-called mesenchymal to epithelial transition. Following publication of this article, Gershongorn retracted this suggestion and showed that it is the mesenchymal stem cells present in the islets that undergo differentiation and not dedifferentiated beta cells [Davani et al., Stem cells, Volume 25, Issue 12, Pages 3215-3222, 2007].
An alternative to forced expansion of post-mitotic β cells is the induction of differentiation of stem/progenitor cells, which have a natural self-expansion capacity, into insulin-producing cells. The directed differentiation of embryonic stem cells has generated cells that only produce low amounts of insulin, compared to β cells, and their potential use in transplantation has met with ethical objections, as well as concerns regarding risk of teratomas.
Adult stem cells have also been differentiated into insulin-producing cells. However, the efficiency of expansion of these cell types in tissue culture and their rate of differentiation into insulin-producing cells need to be greatly improved to allow generation of significant cell numbers for transplantation.
It has been clearly demonstrated that committed cells can be at least partly reprogrammed with dominant genes that activate a cascade of developmental events. U.S. Publication No. 2005/0244966 to the present inventors teaches the reprogramming of fetal hepatic cells into beta-like insulin-producing cells by expression of dominant transcription factors, such as Pdx1, that direct the development of endocrine pancreas. The human fetal liver cells were induced to produce and store mature insulin in significant amounts, about a third of those produced by normal β cells, release it in response to physiological glucose levels, and replace β-cell function in STZ-diabetic nonobese-diabetic severe combined immunodeficient (NOD-scid) mice. The modified cells expressed multiple β-cell genes.
Islet cells have been expanded ex vivo in the presence of epidermal growth factor and nerve growth factor. Although these cells show high insulin content, they do not secrete insulin in response to glucose (Lechner A. et al., Biochem Biophys Res Commun 327:581-588, 2005).
International Application WO2006/054305 teaches expansion of islet cells in CMRL-1066 medium.
A number of factors have been shown to promote both β-cell proliferation and differentiation in tissue culture. Members of the growth hormone family, including placental lactogen (PL), growth hormone (GH) and prolactin (PRL), induce replication in neonatal rat islet cells. Significant mitogenic effects of hepatocyte growth factor (HGF) have been observed on human fetal and adult islets and mouse islets. In the presence of activin A or nicotinamide, HGF has been shown to stimulate β-cell differentiation in cultured fetal pancreatic islets as well as a pancreatic cell line. Glucagon-like peptide 1 (GLP-1), and its more stable analog exendin-4, have been shown to stimulate β-cell proliferation and to induce insulin gene expression in a pancreatic cell line. Members of the epidermal growth factor (EGF) family, including EGF, TGFα and betacellulin, have also been shown to stimulate β-cell proliferation and differentiation. Betacellulin is a potent mitogen for a number of cell types, including islet beta (β) cells. It was shown to increase islet neogenesis in alloxan and STZ-treated mice, and accelerate islet-regeneration in 90%-pancreatectomized rats [Li L, et al., Endocrinology 2001; 142:5379-5385].
Rukstalis et al [Endocrinology 147(6) 2997-3006] teaches that the transcription factor Snail modulates hormone expression in immortalized pancreatic endocrine cell lines. Further, it was disclosed that down-regulation of Snail in those cell lines using siRNA increased insulin gene expression.
U.S. Publication No. 2006/0292127 teaches dedifferentiating, and not redifferentiating, beta cells by contacting the cells with agents that regulate Snail/slug/slit family of transcription factors.
International Application WO2006/054305 teaches redifferentiation of expanded Beta cells in a medium comprising betacellulin and/or ngn-3.